Article pubs.acs.org/ac
Fully Integrated Lab-on-a-Disc for Nucleic Acid Analysis of FoodBorne Pathogens Tae-Hyeong Kim,‡ Juhee Park,‡ Chi-Ju Kim, and Yoon-Kyoung Cho* Department of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), UNIST-gil 50, Ulsan, 689-798, Republic of Korea S Supporting Information *
ABSTRACT: This paper describes a micro total analysis system for molecular analysis of Salmonella, a major food-borne pathogen. We developed a centrifugal microfluidic device, which integrated the three main steps of pathogen detection, DNA extraction, isothermal recombinase polymerase amplification (RPA), and detection, onto a single disc. A single laser diode was utilized for wireless control of valve actuation, cell lysis, and noncontact heating in the isothermal amplification step, thereby yielding a compact and miniaturized system. To achieve high detection sensitivity, rare cells in large volumes of phosphate-buffered saline (PBS) and milk samples were enriched before loading onto the disc by using antibody-coated magnetic beads. The entire procedure, from DNA extraction through to detection, was completed within 30 min in a fully automated fashion. The final detection was carried out using lateral flow strips by direct visual observation; detection limit was 10 cfu/mL and 102 cfu/mL in PBS and milk, respectively. Our device allows rapid molecular diagnostic analysis and does not require specially trained personnel or expensive equipment. Thus, we expect that it would have an array of potential applications, including in the detection of foodborne pathogens, environmental monitoring, and molecular diagnostics in resource-limited settings.
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handling errors, and yield greatly enhanced analytical performances.12,13 Thus, there has been intensive research on the pathogen detection using microfluidic chips.14−19 The Landers group reported a sample-to-answer system for the detection of Bordetella pertussis by integration of solid phase extraction (SPE), PCR, and microchip electrophoresis sequentially.16 The Mathies group and Soper group also developed lab-on-a-chip for pathogen detection employing immune-magnetic cell isolation, electrophoresis, and continuous flow ligase detection reaction.17,19 Even though multifunctions for pathogen detection such as target separation and detection are integrated on a single chip, complex tube connections for pneumatic fluid control are required, which impedes the miniaturization of the full system. Among several lab-on-a-chip platforms, the lab-ona-disc concept has received particular interest owing to the integration of all the required functions onto a disc-shaped device, which enables complete automation of the workflow using only a single rotor.20,21 The centrifugal, inertial, and Coriolis forces generated by the rotation of the disc can be utilized for fluidic pumping,20,22 mixing,23,24 and metering25,26 of reagents for analysis. Moreover, several valving techniques have been developed to manipulate microfluidics on
lthough food safety norms are very rigorous in most economically developed nations, diseases caused by foodborne pathogens continue to cause public health problems worldwide. The potential for an outbreak of diseases caused by these pathogens has also increased owing to the increase in international food trading. Failure to detect pathogenic contamination in food may not only result in large economic losses to the food industry but also could have drastic consequences on human health.1 Conventional methods for detection of food-borne pathogens, which are based on cell cultivation, require at least 3−4 days to yield presumptive results and a maximum of 7 days to yield confirmatory results.2 In order to reduce the analysis time, several methods such as polymerase chain reaction (PCR),3−6 enzyme immunoassay,7−9 fluorescence resonance energy transfer (FRET),2,10 and microarray11 have been developed. However, these techniques require complicated manual steps, specially trained personnel, and expensive equipment. These drawbacks have limited the usage of these techniques in resource-limited environments. Therefore, there is an urgent need for the development of point of care test (POCT) devices to provide rapid, accurate, and automated methods for detection of food-borne pathogens. Micro total analysis systems, also referred to as “lab-on-achip,” are considered strong candidates for POCT devices. Miniaturized size, precise microfluidic control, and automation of the operation enable integration of the necessary processes into a single chip, reduce reagent consumption and manual © 2014 American Chemical Society
Received: December 6, 2013 Accepted: March 17, 2014 Published: March 17, 2014 3841
dx.doi.org/10.1021/ac403971h | Anal. Chem. 2014, 86, 3841−3848
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Article
Figure 1. (a) Expanded view of the lab-on-a-disc showing top and bottom plates made of polycarbonate, strip sensors, adhesive layer, and the metal heater. (b) Top view of a section of the disc featuring the chambers for cell lysis, isothermal amplification, metering, dilution, and detection. (c) Schematic illustration of the experimental setup. Computer-controlled unit includes a spinning motor, a laser for the operation of the ferrowax microvalves, local heating of the amplification chamber, and a strobe light and a CCD camera for the visualization of the fluidic transfer on the rotating disc.
demand.12,25,27 As a result, many biochemical assays requiring multistep processes such as immunoassay, nucleic acid extraction, and blood analysis have been automated on a disc.28−36 Previously, we have demonstrated the extraction of DNA from whole blood spiked with Hepatitis B virus using a lab-ona-disc.31,37 However, in that study, amplification and detection were performed off the chip, using a conventional real-time PCR machine. While PCR utilizes temperature-driven melting and extension of DNA, recombinase polymerase amplification (RPA) is an isothermal DNA amplification technique that uses enzymes to facilitate the binding of oligonucleotide primers to template DNA and to carry out the subsequent elongation step at a constant temperature (between 37 and 40 °C). Although RPA does not require such a finely tuned heating system to maintain a high temperature (95 °C) and the reaction takes only 20 min, the method shows sensitivity similar to that shown by conventional PCR.38 Moreover, RPA provides shorter reaction time at lower temperature compared to other isothermal techniques such as nucleic acid sequence-based amplification (NASBA), loop-mediated amplification (LAMP), strand displacement amplification (SDA), and so on.39,40 Therefore, RPA can be very useful for POCT molecular diagnostic devices, a fact underscored by its use in several microfluidic systems.41−43 For example, Lutz et al.33 developed a disc with prestored lyophilized RPA reagents and detected the antibiotic resistance gene mecA of Staphylococcus aureus using a conventional real-time PCR machine, and Shen et al.34 performed digital RPA on a SlipChip, a microfluidic chip they had developed previously. Both groups used fluorescence detection and confirmed that the performance of RPA was comparable to that of PCR. Furthermore, Rohrman et al.35 demonstrated that lateral flow strips can be used to detect RPA signals, thereby constructing a device that could be used in a resource-limited setting. However, in all of these cases, the DNA extraction step was performed manually and was not integrated with the RPA device. We integrated DNA extraction, RPA, and signal detection onto a single chip. This enabled an entire detection workflow to be performed in less than 30 min. We demonstrate here, the practical utility of this method through the detection of
Salmonella, a major food-borne pathogen, in spiked milk samples.
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EXPERIMENTAL DETAILS Target Concentration. Salmonella enteritidis (KCCM40763, Korean Culture Center of Microorganisms, Korea) was cultured at 37 °C with vigorous aeration in nutrient broth (BD, Franklin Lakes, New Jersey). Then, bacterial cells were harvested by centrifugation, and stock cultures were prepared by resuspending the harvested cells in phosphatebuffered saline (PBS). For the selectivity test, Staphylococcus aureus (ATCC 25923), Enterococcus faecium (ATCC 29212), and Escherichia coli MG 1655 (ATCC 700926) were purchased from American Type Culture Collection (Manassas, VA), and Pseudomonas aeruginosa (KCCM11328) was purchased Korean Culture Center of Microorganisms (Seoul, Korea). All pathogens were prepared by the same procedure as the case for the Salmonella and used for the following experiments. Magnetic beads were coated with anti-Salmonella antibodies (ViroStat, Portland, Maine) as follows. A total of 10 μg of the biotin-modified anti-Salmonella antibodies were added to the 100 μL of prewashed streptavidin-modified magnetic beads (Life Technologies, Oslo, Norway) and incubated for 30 min at room temperature. The tube was placed in a magnet holder for 2 min, and the supernatant was discarded. Finally, the beads were washed twice with 0.1% bovine serum albumin (BSA) in PBS and resuspended in 100 μL of PBS solution. For the target enrichment step, pretreated beads were collected at the bottom of the tube with a magnet holder, and PBS solution was removed. Then the beads were resuspended in 1 mL of 101∼106 cfu/mL of Salmonella-spiked PBS or milk and incubated at room temperature with gentle agitation. After incubation for 10 min, the supernatant was discarded and the beads were washed twice with 0.05% Tween 20 in PBS and resuspended in 5 μL of distilled water. The capture efficiency of the antibody-coated magnetic beads was determined using a real-time PCR kit (Kogen Biotech, Seoul, Korea) with a thermal cell lysis step in which the sample was kept at 95 °C for 10 min in a heating block. RPA. For the final Salmonella detection, we used a commercially available lateral flow-type RPA kit (TwistDX, Cambridge, U.K.) consisting of the dried reagent, the 3842
dx.doi.org/10.1021/ac403971h | Anal. Chem. 2014, 86, 3841−3848
Analytical Chemistry
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Figure 2. The effects of the key variables for the pre-enrichment step are investigated by real time PCR. (a) The bead concentration in the sample solution for the binding reaction of the target pathogen and the antibody-coated magnetic beads, (b) the incubation time, and (c) sample type, PBS or milk, spiked with Salmonella at different concentrations were tested. (d) The efficiency of the laser lysis was compared with standard thermal lysis and (e) the effect of the laser irradiation time was investigated.
sensor chamber before assembling the top and bottom plates. Finally, the aluminum plates (10 mm × 10 mm) were inserted under the amplification chambers and utilized for local temperature control. Figure 1c presents the operation system of the disc; for a detailed description of the setup, readers should refer to a previous report.28,44 Briefly, a strobe lamp (B&B Corporation, Seoul, Korea) and a CCD camera (Toshiba Corp., Tokyo, Japan) were used to visualize the rotating disc in real time, and a laser diode (Associated Opto-Electronics Corp., Chongqing, China) was installed for the actuation of the ferrrowax microvalves. In addition, the diode was utilized for the local heating of the liquid sample for the DNA extraction as well as for isothermal amplification.
rehydration buffer, Mg(OAc)2 solution as an initiator, lateral flow strips, and PBS with Tween 20 (PBST). Dried reagent in a tube was gently mixed with 37.5 μL of the rehydration buffer. Half of the rehydrated RPA reagent was preloaded into the amplification chamber, and 1.25 μL of Mg(OAc)2 solution was introduced into the lysis chamber together in order to prevent aberrant initiation of amplification step. After DNA extraction, the DNA solution was moved to the amplification chamber and mixed with preloaded RPA reagents. RPA was performed at 37−40 °C for 20 min, which is the recommended condition for this method. A volume of 10 μL of the resulting amplicon was mixed with 100 μL of PBST and was absorbed onto lateral flow strips, which comprise a conjugation pad, composed of gold nanoparticles conjugated with anti-FITC antibodies and a test line, containing immobilized antibiotin antibodies. RPA products are labeled with a 5′-FITC antigen and 3′-biotin, which facilitates binding to the gold nanoparticles and the test line in a sandwich format. In addition, anti-rabbit antibodies coated on a control line were used to capture gold nanoparticles as an internal technical control. The test for the final confirmation of the result was conducted 5 min after absorption of the sample solution. Disc Fabrication. A lab-on-a-disc for fully automated Salmonella detection was designed as shown in Figure 1. The main body of the disc was composed of top and bottom polycarbonate layers, which were bonded with a pressuresensitive adhesive (FLEXcon, Spencer, MA) (Figure 1a). The channels and chambers were designed using 3D autoCAD program (details in Figure 1b). The fluidic chambers for cell lysis, isothermal amplification, metering, dilution, and detection were located at the bottom of the disc, and the reverse side of the amplification chamber was carved for the insertion of a metal plate. The ferrowax microvalves were located at the top of the disc and were actuated on demand by laser irradiation.25 The disc was fabricated through computer numerical control milling, and the lateral flow strips were inserted into the strip
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RESULTS AND DISCUSSION For sensitive detection of food-borne pathogens, most analysis protocols employ either traditional culture or rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and PCR-based assays, which in turn require a pre-enrichment step. In our system, selective enrichment of the target from a large volume of the sample is achieved through immunomagnetic separation. Even though it is imperative to have large volume process capability for a highly sensitive rare cell detection technology, it still remains as an unmet need for microfluidic chips. In this work, rare cells in large volumes of PBS and milk samples were manually enriched before loading onto the disc by using antibody-coated magnetic beads. However, our enrichment method can realize larger volume treatment easily by repeating sample incubation and removal of supernatant several times. The capture efficiency of the immunomagnetic separation step was determined using real-time PCR. Briefly, cells in 1 mL of PBS or milk spiked with Salmonella (101 to 104 cfu/mL) were enriched to 5 μL of solution containing magnetic beads and subjected to tabletop thermal lysis, and the resultant solutions were used for real-time PCR. The number of bacteria 3843
dx.doi.org/10.1021/ac403971h | Anal. Chem. 2014, 86, 3841−3848
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Figure 3. (a) Detailed schematic of indirect heating system we tested and (b) plot presenting the temperature profile of metal plate, sample solution in amplification chamber with heating using laser irradiation (black line), and conventional peltier type thermoblock (blue dot line). Simulated temperature profile of a disc as laser is irradiated to heat solution at 40 °C: (c) top surface of a disc, (d) cross sectional profile near the heating zone and (e,f) linear profiles along the dot lines, A−B and C−D, respectively. Zero point of parts e and f) are the center of the metal plate and the top surface of the bonded chip, respectively.
inhibit the binding of target pathogens to the magnetic beads. However, the best capture efficiency reported for the immunomagnetic separation of bacteria from real samples was lower than 20%, suggesting that our experimental conditions are relatively well optimized.45,46 In previous studies, a fast and efficient DNA extraction method was developed using laser irradiation on magnetic particles to capture target bacteria.31,37 To compare the efficiency of the proposed laser lysis method with that of a conventional thermal lysis method, the cells were lysed by heating the samples at 95 °C for 10 min in a heating block, and the lysate was analyzed with real-time PCR. When evaluating cell lysis conditions, we found that laser irradiation for at least 5 s was as effective as thermal lysis for 10 min (Figure 2d). However, to ensure complete lysis in the following experiments, we laser irradiated the samples for 20 s. We next evaluated the cell lysis obtained for the samples at various concentrations (Figure 2e). A 5 μL of a solution containing pathogens captured on magnetic beads enriched from PBS (1 mL) spiked with Salmonella at various concentrations ranging from 101 to 104 cfu/mL was introduced into the lysis chambers on the disc. Following 20 s laser irradiation for cell lysis, the lysates were collected from the lysis chamber and were subjected to real-time PCR to quantify the sample DNA concentration. The results show that the laser lysis method has comparable lysis efficiency to that of standard lysis techniques at all the tested concentrations. Further, we introduced a laser-assisted indirect heating system for isothermal DNA amplification and evaluated the heating performance over 20 min. In previous studies, infrared light based wireless but direct heating methods were explored for biosensing applications.14,16 Here, one laser diode (808 nm) was used not only for the actuation of the ferrowax valves but also for the local and wireless heating for the isothermal amplification. Figure 3a shows the detailed system for the laser-
was calculated using the calibration curve generated using Salmonella suspensions of known concentrations (100−107 cfu/ mL). Finally, the capture efficiency was calculated using the following equation: capture efficiency(%) no. of bacteria in enriched sample = × 100% no. of bacteria in input sample
We optimized several critical experimental variables for the immonomagnetic enrichment step (Figure 2). First, the effect of the bead concentration on the capture efficiency was evaluated. The maximum capture efficiency was observed at a bead concentration >1 mg/mL, i.e., 100 μL of C1 bead stock solution (10 mg/mL) per assay (1 mL of sample), Figure 2a. The incubation time of pathogen capture by the antibodycoated beads was also investigated. As shown in Figure 2b, the capture efficiency reached the maximum from 10 min of incubation. On the basis of these optimization experiments, we introduced the Salmonella-specific binding antibody-coated magnetic beads (1 mg) into the test sample (1 mL) and incubated for 10 min for the target enrichment step. Then, the magnetic beads were resuspended in 5 μL of distilled water after removing the supernatant. This yielded 5 μL of 200-foldenriched sample solution, which was used for further analysis. Figure 2c shows the capture efficiency for 1 mL of PBS or milk spiked with Salmonella with the concentration ranging from 101 to 104 cfu/mL. In the case of Salmonella-spiked PBS, more than 90% capture efficiency was observed for 102−104 cfu/mL solutions, while the efficiency was as low as 43% for 10 cfu/mL samples. On the contrary, Salmonella in milk was captured with relatively low efficiency (
